The authors acknowledge Dr. Luk H. Vandenberghe (Harvard Medical School, Department of Ophthalmology) for providing purified AAV-3. This work was supported by the National Institutes of Health and the National Cancer Institute (R01CA193578, R01CA227261, R01CA219700 to D.F.K.).

1. Loading the specimen holder for liquid-EM
<ol>
	<li>Clean the silicon nitride (SiN) microchips by incubating each chip in 150 mL of acetone for 2 min followed by incubation in 150 mL of methanol for 2 min. Allow chips to dry in laminar airflow.</li>
	<li>Plasma clean the dried chips using a glow-discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.</li>
	<li>Load a dry base microchip into the tip of the specimen holder. Add ~0.2 &#181;L of sample (0.2-1 mg/mL of virus assemblies in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl2; 10 mM CaCl2) to the base chip. Following a 1-2 min incubation step, place the top chip on the wet base chip containing the sample.</li>
	<li>Clamp the assembly together to form a hermetically sealed enclosure, held in place mechanically by three brass screws. Upon sealing the assembly, pump the tip to 10-6 Torr using a turbo-pumped dry pumping station. The holder is now ready to be inserted into the TEM. 
	NOTE: The select holder has no cooling capabilities and is not used for cryo-EM.</li>
</ol>
2. Production of microchip sandwich assemblies
NOTE: Different SiN or silicon dioxide (SiO) microchips can be used directly from the shipped gel packs. Carbon-coated gold grids may also be used directly as supplied.
<ol>
	<li>Plasma clean the microchips and carbon grids using a glow discharge instrument operating under standard conditions of 30 W, 15 mA for 45 s using Argon gas.</li>
	<li>Add ~2 &#181;L of sample contained in 50 mM HEPES, pH 7.5; 150 mM NaCl; 10 mM MgCl2; 10 mM CaCl2&#160;to a glow-discharged microchip placed on a gel pack. Remove excess solution (~50%) using a filter paper or a pipette. Following a 1-2 min incubation step, add the glow-discharged carbon grid to the wet microchip containing the sample.</li>
	<li>Clamp the assembly together using a single-tilt specimen holder or autoloader grid clips at room temperature to form a hermetically sealed enclosure. For the auto-loader clamps, place the sandwich assembly on the bottom C-clip, place the top clip on top of the assembly and use the standard clamping tool to seal the assembly together. 
	NOTE: The clamping procedure performed here uses the same steps as for cryo-EM grid clamping, but at room temperature. Clamped samples may be stored for 2 months or longer prior to imaging while maintaining liquid in the enclosure. No leak check is used with a room temperature specimen holder or the autoloader.</li>
	<li>The specimen is now ready to be inserted into the TEM. Examine samples placed in autoloaders under cryo conditions or at room temperature.</li>
</ol>
3. Imaging specimens using a transmission electron microscope
<ol>
	<li>Liquid-EM imaging
	<ol>
		<li>Load the&#160;specimen holder into the TEM equipped with a field-emission gun (FEG) and operating at 200 kV.</li>
		<li>Turn on the gun and adjust the eucentric height of the microscope stage with respect to the specimen by using the wobbler function, tilting the sample from -15&#176; to +15&#176; in the column. This procedure adjusts the stage in the Z-direction to accommodate the sample thickness. And helps ensure an accurate magnification during image recording.</li>
		<li>Record images as long-framed movies using the in situ package integrated with a direct detector having a pixel spacing of 6 &#181;m (Video 1&#160;and Video 2). Individual images may also be recorded using the serial data collection software package20, implementing automated imaging routines. Acquire images under low-dose conditions at magnifications ranging from 28,000x-92,000x and 40 frames per second.</li>
		<li>Adjust exposure times (0.25-1 s) to minimize beam damage to the specimen. Use a defocus range of -1-4 &#181;m at the specified magnification. If a thick solution is encountered, use higher defocus values or select a different region of interest.</li>
		<li>Ensure the solution is present in the samples throughout the imaging session by focusing the electron beam on a sacrificial area not used for data collection, until bubbles are formed (Supplementary Figure 1).</li>
	</ol>
	</li>
	<li>Cryo-EM imaging
	<ol>
		<li>Load the&#160;clipped EM grids or microchip sandwiches into the TEM equipped with a FEG and operating at 300 kV. Turn on the gun and adjust the eucentric height of the microscope stage, using a similar procedure described for the liquid-TEM above (step 3.1.2).</li>
		<li>Record individual images using the single particle analysis system integrated within the microscope system while implementing automated imaging routines. Record images under low-dose conditions using the single particle analysis direct electron detector having a pixel spacing of 14 &#181;m at a magnification of 59,000x and 40 frames per second.</li>
		<li>Use a defocus range of 1-4 &#181;m at the specified magnification. If thick layers of vitreous ice are encountered, use higher defocus values, or select a different region for data collection.</li>
	</ol>
	</li>
</ol>
4. Data analysis and 3 D structure comparisons
<ol>
	<li>Analysis of adeno-associated virus (AAV) in liquid and vitreous ice
	<ol>
		<li>Process movies for AAV particles in liquid and ice using RELION-3.08 program21 or other image processing software22. Perform motion correction using MotionCor2 v1.2.3.</li>
		<li>Once corrected, extract particles using the auto-picking tool in the program software package. Typical box sizes are 330 pixels for liquid specimens and 350 pixels for ice specimens.</li>
		<li>Calculate initial reconstructions using C1 symmetry using the program&#39;s 3D initial model routine and/or the ab-initio model options in the data processing software package. In the program, use a regularization parameter of T = 4 and a pixel size of 1.01 A&#778; for liquid specimens and 1.13 A&#778; for ice specimens.</li>
		<li>Use a mask value of 300 A&#778; throughout the refinement procedures. Perform refinement protocols in the data processing software using I1 symmetry to obtain multiple EM maps. Expected structural resolution may be in the range of 4 A&#778; or better. Use particle equivalency implementing icosahedral symmetry at 16,800 for liquid and 15,240 for ice12,14. 
		NOTE: Resolution estimates are based on the gold-standard Fourier shell correlation (FSC) criteria.</li>
		<li>Mask EM maps at ~250 A&#778; and examine results using a molecular structure analysis software package23,24. In Figure 3, slices are shown in increments of ~5 nm.</li>
		<li>Extract capsid protein (VP1) subunits from the EM maps for comparison. Quantify dynamic changes among EM structures based on the particle diameter measurements (Supplementary Figure 2), and then visualize using the Morph map function in the software.</li>
	</ol>
	</li>
	<li>Analysis of SARS-CoV-2 sub-viral assemblies in liquid
	<ol>
		<li>Process movies using the RELION-3.08 program. Correct for image drift and beam-induced motion using MotionCor2 v1.2.3. Correct for the contrast transfer function (CTF) of the microscope.</li>
		<li>Use the auto-picking tool in the program to select viral assemblies with a box size of 800 pixels. For computational efficiency, extracted particles can be rescaled to 256 pixels. Obtain an initial model using C1 symmetry in the program with a regularization parameter of T = 2&#160;and 1.66 A&#778; pixel size.</li>
		<li>Perform 3D refinement in the program to obtain an EM map, an example is shown at ~8.25 A&#778; according to standard FSC criteria (Figure 4). Visualize EM maps using the molecular structure analysis software with slices incremented at 25 nm as demonstrated in Figure 4.</li>
	</ol>
	</li>
	<li>Analysis of rotavirus double-layered particles (DLPs) in vitreous ice
	<ol>
		<li>Process movies for rotavirus DLPs using other image processing software. Use MotionCor2 v1.2.3 to correct for drift in the images. Use Patch CTF estimation to correct for lens effects on the images.</li>
		<li>Extract particles using the auto-picking tool in the software with a box size of 950 pixels. Down-sample the box size as needed for computing purposes.</li>
		<li>Calculate initial models using ab-initio options and C1 symmetry. Use refinement parameters including a pixel size of 1.47 A&#778; and a mask value of 800 A&#778;.</li>
		<li>Perform additional refinement routines while imposing I1 symmetry. Example results include a 10.15 A&#778; EM map, according to the gold-standard FSC criteria. Total particles used were 2050, which equates to 123,000 protomer units due to the icosahedral symmetry operator (Figure 5).</li>
		<li>Mask final maps at ~750 A&#778; and visualize results in the molecular structure analysis software package. Example slices through the EM map are shown in Figure 5 in increments of ~10 nm.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Deng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly,+structure,+function+and+regulation+of+type+III+secretion+systems.">Assembly, structure, function and regulation of type III secretion systems.</a> Nature Reviews Microbiology. 15 (6), 323-337 (2017).</li><li>Oikonomou, C. M., Chang, Y. -. W., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+view+into+prokaryotic+cell+biology+from+electron+cryotomography.">A new view into prokaryotic cell biology from electron cryotomography.</a> Nature Reviews Microbiology. 14 (4), 205-220 (2016).</li><li>Murata, K., Wolf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-electron+microscopy+for+structural+analysis+of+dynamic+biological+macromolecules.">Cryo-electron microscopy for structural analysis of dynamic biological macromolecules.</a> Biochimica et Biophysica Acta. General Subjects. 1862 (2), 324-334 (2018).</li><li>DiMaio, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+accuracy+models+from+4.5+&#197;+cryo-electron+microscopy+data+with+density-guided+iterative+local+refinement.">Atomic accuracy models from 4.5 &#197; cryo-electron microscopy data with density-guided iterative local refinement.</a> Nature Methods. 12 (4), 361-365 (2015).</li><li>Frank, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+protein+synthesis+based+on+cryo-electron+microscopy+of+the+E.+coli+ribosome.">A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome.</a> Nature. 376 (6539), 441-444 (1995).</li><li>Dukes, M. J., Gilmore, B. L., Tanner, J. R., McDonald, S. M., Kelly, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+TEM+of+biological+assemblies+in+liquid.">In situ TEM of biological assemblies in liquid.</a> Journal of Visualized Experiments. (82), e50936 (2013).</li><li>Dearnaley, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid-cell+electron+tomography+of+biological+systems.">Liquid-cell electron tomography of biological systems.</a> Nano Letters. 19 (10), 6734-6741 (2019).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+wet+biological+samples+by+graphene+liquid+cell+transmission+electron+microscopy.">Direct observation of wet biological samples by graphene liquid cell transmission electron microscopy.</a> Nano Letters. 15 (7), 4737-4744 (2015).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Motion+of+DNA-Au+nanoconjugates+in+graphene+liquid+cell+electron+microscopy.">3D Motion of DNA-Au nanoconjugates in graphene liquid cell electron microscopy.</a> Nano Letters. 13 (9), 4556-4561 (2013).</li><li>Yuk, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+EM+of+colloidal+nanocrystal+growth+using+graphene+liquid+cells.">High-resolution EM of colloidal nanocrystal growth using graphene liquid cells.</a> Science. 336 (6077), 61-64 (2012).</li><li>Wang, X., Yang, J., Andrei, C. M., Soleymani, L., Grandfield, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+of+calcium+phosphate+revealed+by+in+situ+liquid-phase+electron+microscopy.">Biomineralization of calcium phosphate revealed by in situ liquid-phase electron microscopy.</a> Communications Chemistry. 1, 80 (2018).</li><li>Jonaid, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+human+viruses+in+liquid+droplets.">High-resolution imaging of human viruses in liquid droplets.</a> Advanced Materials. 33 (37), 2103221 (2021).</li><li>Pohlmann, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+visualization+of+nanoparticles+interacting+with+glioblastoma+stem+cells.">Real-time visualization of nanoparticles interacting with glioblastoma stem cells.</a> Nano Letters. 15 (4), 2329-2335 (2015).</li><li>Jonaid, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tools+to+advance+high-resolution+imaging+in+liquid.">Automated tools to advance high-resolution imaging in liquid.</a> Microscopy and Microanalysis. , 1-10 (2022).</li><li>Varano, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customizable+cryo-EM+chips+improve+3D+analysis+of+macromolecules.">Customizable cryo-EM chips improve 3D analysis of macromolecules.</a> Microscopy and Microanalysis. 25, 1310-1311 (2019).</li><li>Alden, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM-on-a-chip:+Custom-designed+substrates+for+the+3D+analysis+of+macromolecules.">Cryo-EM-on-a-chip: Custom-designed substrates for the 3D analysis of macromolecules.</a> Small. 15 (21), 1900918 (2019).</li><li>Tanner, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-SiN+&#8211;+An+alternative+substrate+to+visualize+active+viral+assemblies.">Cryo-SiN &#8211; An alternative substrate to visualize active viral assemblies.</a> Journal of Analytical and Molecular Techniques. , (2016).</li><li>Solares, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchip-based+structure+determination+of+disease-relevant+p53.">Microchip-based structure determination of disease-relevant p53.</a> Analytical Chemistry. 92 (23), 15558-15564 (2020).</li><li>Casasanta, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchip-based+structure+determination+of+low-molecular+weight+proteins+using+cryo-electron+microscopy.">Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.</a> Nanoscale. 13 (15), 7285-7293 (2021).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+data+acquisition+from+electron+microscopes+with+SerialEM.">Advanced data acquisition from electron microscopes with SerialEM.</a> Microscopy and Microanalysis. 24, 864-865 (2018).</li><li>Scheres, S. H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RELION:+Implementation+of+a+Bayesian+approach+to+cryo-EM+structure+determination.">RELION: Implementation of a Bayesian approach to cryo-EM structure determination.</a> Journal of Structural Biology. 180 (3), 519-530 (2012).</li><li>Punjani, A., Rubinstein, J. L., Fleet, D. J., Brubaker, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cryoSPARC:+algorithms+for+rapid+unsupervised+cryo-EM+structure+determination.">cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination.</a> Nature Methods. 14 (3), 290-296 (2017).</li><li>Pettersen, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCSF+Chimera&#8212;A+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera&#8212;A visualization system for exploratory research and analysis.</a> Journal of Computational Chemistry. 25 (13), 1605-1612 (2004).</li><li>Goddard, T. D., Huang, C. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+density+maps+with+UCSF+Chimera.">Visualizing density maps with UCSF Chimera.</a> Journal of Structural Biology. 157 (1), 281-287 (2007).</li><li>Lerch, T. F., Xie, Q., Chapman, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+adeno-associated+virus+serotype+3B+(AAV-3B):+Insights+into+receptor+binding+and+immune+evasion.">The structure of adeno-associated virus serotype 3B (AAV-3B): Insights into receptor binding and immune evasion.</a> Virology. 403 (1), 26-36 (2010).</li><li>Sharma, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+grid-based+cryo-EM+of+PKC+binding+to+RACK1+on+the+ribosome.">Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.</a> Journal of Structural Biology. 181 (2), 190-194 (2013).</li><li>Kiss, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+enveloped+viruses+on+affinity+grids+for+downstream+cryo-electron+microscopy+applications.">Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications.</a> Microscopy and Microanalysis. 20 (1), 164-174 (2014).</li><li>Degen, K., Dukes, M., Tanner, J. R., Kelly, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+affinity+capture+devices&#8212;a+nanoscale+purification+platform+for+biological+in+situ+transmission+electron+microscopy.">The development of affinity capture devices&#8212;a nanoscale purification platform for biological in situ transmission electron microscopy.</a> Rsc Advances. 2 (6), 2408-2412 (2012).</li><li>Hui, S. W., Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+biological+membranes.">Electron diffraction of wet biological membranes.</a> Science. 184 (4132), 77-78 (1974).</li><li>Hui, S. W., Parsons, D. F., Cowden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+phospholipid+bilayers.">Electron diffraction of wet phospholipid bilayers.</a> Proceedings of the National Academy of Sciences of the United States of America. 71 (12), 5068-5072 (1974).</li><li>Parsons, D. F., Matricardi, V. R., Moretz, R. C., Turner, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+and+diffraction+of+wet+unstained+and+unfixed+biological+object.">Electron microscopy and diffraction of wet unstained and unfixed biological object.</a> Advances in Biological and Medical Physics. 15, 161-270 (1974).</li><li>Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+wet+specimens+in+electron+microscopy.+Improved+environmental+chambers+make+it+possible+to+examine+wet+specimens+easily.">Structure of wet specimens in electron microscopy. Improved environmental chambers make it possible to examine wet specimens easily.</a> Science. 186 (4162), 407-414 (1974).</li><li>Matricardi, V. R., Moretz, R. C., Parsons, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+diffraction+of+wet+proteins:+Catalase.">Electron diffraction of wet proteins: Catalase.</a> Science. 177 (4045), 268-270 (1972).</li></ol>

The authors declare that they have no competing financial interests. The author, Madeline J. Dressel-Dukes, is an employee of Protochips, Inc. and Michael Spilman is an employee of DirectElectron.

New opportunities are presented to streamline current liquid-EM workflows by using new automated tools and technologies adapted from the cryo-EM field. Applications involving the new microchip sandwich technique are significant with respect to other methods because they enable high-resolution imaging analysis in liquid or vitreous ice. One of the most critical steps in the protocol is producing specimens with the ideal liquid thickness to visualize exquisite details at the nanoscale level. Ideal regions of interest are i...

Biomedical research improves our understanding of human health and disease through the development of new technologies. High-resolution imaging is transforming our view of the nanoworld - permitting us to study cells and molecules in exquisite detail1,2,3,4,5. Static information of dynamic components such as soft polymers, protein assemblies, or human viruses reveals only a limited snapshot of their complex narrative. To better understand how molecular entities operate, their structure and function must be jointly investigated.
Recent advances in the production of materials such as atomically thin graphene&#160;or silicon-based microchips provide new opportunities for real-time structure-function analysis using transmission electron microscopes (TEMs). These materials can create hermetically sealed chambers for live EM imaging6,7,8,9,10,11. The new field of liquid-EM, the room temperature correlate to cryo-EM, provides unprecedented views of hard or soft materials in solution, allowing scientists to simultaneously study the structure and dynamics of their specimen. Liquid-EM applications include real-time recordings of therapeutic nanoparticles interacting with cancer stem cells as well as changes in the molecular intricacies of viral pathogens12,13,14.
Just as methodological advances spurred the resolution revolution in the cryo-EM field, new techniques and methods are needed to extend the use of liquid-EM as a high-throughput tool for the scientific community. The overall goal of the methods presented here is to streamline liquid-EM specimen preparation protocols. The rationale behind the developed techniques is to employ new microchip designs and autoloader devices, suitable for both liquid- and cryo-EM data collection (Figure 1)7,14,15,16,17. The assemblies are mechanically sealed using standard grid clips for automated instruments, such as the Krios, which can accommodate multiple samples per session or a F200C TEM (Figure 2). This methodology expands the use of high-resolution imaging beyond standard cryo-EM applications demonstrating broader purposes for real-time materials analysis.
In the current video article, protocols are presented for preparing virus assemblies in liquid with and without commercially available specimen holders. Using the specialized specimen holder for liquid-EM, thin liquid specimens can provide structural information comparable to cryo-EM samples, as well as dynamic insights of the specimens. Also demonstrated are methods for preparing liquid specimens using autoloader tools for high-throughput routines. The major advantage over other techniques is that automated specimen production allows the user to quickly assess their samples for optimum thickness and electron dosage prior to data collection. This screening technique quickly identifies ideal areas for real-time recordings in liquid or ice12,14,18,19. For purposes of 3D structure determination, liquid-EM may complement the long-established cryo-EM methods implemented in cryo-EM. Readers employing conventional TEM or cryo-EM technologies may consider using liquid-EM workflows to provide new, dynamic observations of their samples in a manner that complements their current strategies.
Virus samples used in this protocol include purified adeno-associated virus subtype 3 (AAV) obtained as a gift and cultured under standard conditions12. Also used were non-infectious SARS CoV-2 sub-viral assemblies derived from the serum of COVID-19 patients12&#160;and obtained from a commercial source. Finally, purified simian rotavirus (SA11 strain) double-layered particles (DLPs) were obtained from the laboratory of Dr. Sarah M. McDonald Esstman at Wake Forest University and cultured using standard conditions 6,17. Software packages described here are freely available and the links have been provided in the Table of Materials section.

<table><tbody><tr><td>Acetone</td><td>Fisher Scientific</td><td>&amp;nbsp;A11-1</td><td>1 Liter</td></tr><tr><td>Autoloader clipping tool</td><td>ThermoFisher Scientific</td><td>N/A</td><td>Also SubAngstrom supplier</td></tr><tr><td>Autoloader grid clips</td><td>ThermoFisher Scientific</td><td>N/A</td><td>top and bottom clips</td></tr><tr><td>Carbon-coated gold EM grids</td><td>Electron Microcopy Sciences</td><td>CF400-AU-50</td><td>400-mesh, 5-nm thickness</td></tr><tr><td>COVID-19 patient serum</td><td>RayBiotech</td><td>CoV-Pos-S-500</td><td>500 microliters of PCR+ serum</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>&amp;nbsp;A412-1</td><td>1 Liter</td></tr><tr><td>Microwell-integrad microchips</td><td>Protochips, Inc.</td><td>EPB-42A1-10</td><td>10x10-mm window arrays</td></tr><tr><td>TEMWindows microchips</td><td>Simpore Inc.</td><td>SN100-A10Q33B</td><td>9 large windows, 10-nn thick</td></tr><tr><td>TEMWindows microchips</td><td>Simpore, Inc.&amp;nbsp;</td><td>SN100-A05Q33A</td><td>9 small windows, 5-nm thick</td></tr><tr><td>Top microchips</td><td>Protochips, Inc.</td><td>EPT-50W</td><td>500 mm x 100 mm window</td></tr><tr><td>Whatman #1 filter paper</td><td>Whatman</td><td>1001 090</td><td>100 pieces, 90 mm</td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DirectView direct electron detector</td><td>Direct Electron</td><td></td><td>6-micron pixel spacing</td></tr><tr><td>Falcon 3 EC direct electron detector</td><td>ThermoFisher Scientific</td><td></td><td>14-micron pixel spacing</td></tr><tr><td>Gatan 655 Dry pump station</td><td>Gatan, Inc.</td><td></td><td>&amp;nbsp;Pump holder tip to 10&lt;sup&gt;-6 &lt;/sup&gt;range</td></tr><tr><td>Mark IV Vitrobot</td><td>ThermoFisher Scientific</td><td></td><td>state-of-the-art specimen preparation unit&amp;nbsp;</td></tr><tr><td>PELCO easiGlow, glow discharge unit</td><td>Ted Pella, Inc.</td><td></td><td>&amp;nbsp;Negative polarity mode</td></tr><tr><td>Poseidon Select specimen holder</td><td>Protochips, Inc.</td><td></td><td>&amp;nbsp;FEI compatible;specimen holder</td></tr><tr><td>Talos F200C TEM</td><td>ThermoFisher Scientific</td><td></td><td>200 kV; Liquid-TEM</td></tr><tr><td>Titan Krios G3</td><td>ThermoFisher Scientific</td><td></td><td>300 kV; Cryo-TEM</td></tr><tr><td>&lt;strong&gt;Freely available software&lt;/strong&gt;</td><td>&lt;strong&gt;Website link&lt;/strong&gt;</td><td></td><td>&lt;strong&gt;Comments (optional)&lt;/strong&gt;</td></tr><tr><td>cryoSPARC</td><td>https://cryosparc.com/</td><td></td><td>other image processing software</td></tr><tr><td>CTFFIND4</td><td>https://grigoriefflab.umassmed.edu/ctffind4</td><td></td><td>CTF finding program</td></tr><tr><td>MotionCorr2</td><td>https://emcore.ucsf.edu/ucsf-software</td><td></td><td></td></tr><tr><td>RELION</td><td>https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page</td><td></td><td></td></tr><tr><td>SerialEM</td><td>https://bio3d.colorado.edu/SerialEM/</td><td></td><td></td></tr><tr><td>UCSF Chimera</td><td>https://www.cgl.ucsf.edu/chimera/</td><td></td><td>molecular structure analysis software package</td></tr></tbody></table>

advancing high-resolution imaging of virus assemblies in liquid and ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

A liquid-TEM operating at 200 kV was used for all liquid-EM imaging experiments and a cryo-TEM operating at 300 kV was used for all cryo-EM data collection. Representative images and structures of multiple viruses are presented to demonstrate the utility of the methods across various test subjects. These include recombinant adeno-associated virus subtype 3 (AAV), SARS-CoV-2 sub-viral assemblies derived from the patient serum, and simian rotavirus double-layered particles (DLPs), SA11 strain. First, comparisons are demons...

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Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

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Research

JoVE Journal

Biochemistry

2.9K Views.  Pennsylvania State University. De belangstelling voor vloeibare elektronenmicroscopie is de afgelopen jaren omhooggeschoten, omdat we nu realtime processen op nanoschaal kunnen visualiseren. Verbeterde kennis over deze flexibele structuren kan ons helpen bij het ontwikkelen van nieuwe reagentia om opkomende pathogenen zoals SARS-CoV-2 te bestrijden. Het bekijken van eiwitten in een vloeibare omgeving helpt om biologische systemen na te bootsen en geeft ons de mogelijkheid om op een meer dynamische manier naar de eiwitten t...

Video: Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Fabrication of Micro-Patterned Chip with Controlled Thickness for High-Throughput Cryogenic Electron Microscopy

A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the difficulty of preparing cryo-EM samples with controlled ice thickness at the nanoscale. The silicon (Si)-based chip, which has a regular array of micro-holes with graphene oxide (GO) window patterned on a thickness-controlled silicon nitride (SixNy) film, has been developed by applying microelectromechanical system (MEMS) techniques. UV photolithography, chemical vapor deposition, wet and dry etching of the thin film, and drop-casting of 2D nanosheet materials were used for mass-production of the micro-patterned chips with GO windows. The depth of the micro-holes is regulated to control the ice thickness on-demand, depending on the size of the specimen for cryo-EM analysis. The favorable affinity of GO toward biomolecules concentrates the biomolecules of interest within the micro-hole during cryo-EM sample preparation. The micro-patterned chip with GO windows enables high-throughput cryo-EM imaging of various biological molecules, as well as inorganic nanomaterials.

A newly developed micro-patterned chip with graphene oxide windows is fabricated by applying microelectromechanical system techniques, enabling efficient and high-throughput cryogenic electron microscopy imaging of various biomolecules and nanomaterials.

A major limitation for the efficient and high-throughput structure analysis of biomolecules using cryogenic electron microscopy (cryo-EM) is the ...

Engineering

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Neuroscience

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Biology

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

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